Method of producing porous spherical particles

ABSTRACT

The invention refers to a method of controlling the porosity of porous spherical particles produced from a polysaccharide dissolved in a solvent, in which it can be gelled. The polysaccharide solution is finely divided by mechanical means into spherical droplets which are allowed to pass through a humid atmosphere and transferred to a capturing medium while controlling the temperature and humidity of humid atmosphere.

The present invention refers to a method of producing porous sphericalgelled particles. More precisely, the invention refers to a method ofcontrolling the porosity of porous spherical particles produced from apolysaccharide dissolved in a solvent, in which it can be gelled, saidpolysaccharide solution being finely divided by mechanical means intospherical droplets which are allowed to pass through a humid atmosphereand transferred to a capturing medium.

Gel filtration is a commonly used chromatographic separation method inwhich molecules are separated with respect to their size. Smallmolecules diffuse into porous particles while large molecules pass onthe outside thereof. Spherical particles intended for the separation ofmacromolecules by means of such a chromatography are produced from agelled and/or polymerized material. Of course, a good separation doesnot only depend on the size and the size distribution of the particlesbut also on the extent of the porosity of the particles.

In this connection the porosity of porous particles refers to theapparent size exclusion dimensions of a porous matrix as described byHagel et al. (“Apparent Pore-Size Distributions of ChromatographyMedia”, J. Chromatogr. 743(1):33-42, 1996).

Porous matrices, which are used for gel filtration, can after one orseveral modifications with advantage also be used for other technologiesincluding chromatographic separations. In these technologies, e.g. ionexchange chromatography (IEX), hydrophobic interaction chromatography(HIC), affinity chromatography, reversed phase chromatography etc,various interactions with direct and/or substituted ligand(s) on the gelare utilized, which are also highly dependent on the porosity. Suchmatrices can also be used for controlled release in connection with drugdelivery.

Normally, these particles are produced by means of dispersion procedureswhich are based on a vigorous mechanical mixing of the generally watersoluble substance which is to be dispersed in a dispersing mediumusually comprising an organic solvent such as toluene. The processing ofthe spherical particles obtained from the dispersing procedure takesplace in several steps accompanied by a thorough rinsing in order toremove the solvent. This conventional method of producing the particleshaped material for chromatography is thus energy demanding as well asexpensive and, in addition, the productivity is low and producedbatchwise.

One way of avoiding organic solvents when producing such particles wouldbe to use water as a solvent in the manufacturing procedure. Forexample, a method for producing spherical alginate particles is shown inthe publication WO 93/02785, the alginate solution being finely dividedinto droplets which are allowed to fall freely through the air down intoan ionic solution in which they are hardened.

In experiments of producing particles from water soluble polysaccharidesattempts have been made to allow droplets of a polysaccharide solutionto gel directly into particles in water of 0-25° C. The results obtainedin this connection have, however, not been satisfactory with referenceto such problems as shape, gel concentration and surface faceproperties.

It is thus important that droplets of uniform size are produced. In WO95/20620 spherical gel particles for chromatographic use are produced bymechanical disintegration of a gelling liquid polymer and transport ofthe particles through a gas to a solvent. In this way, particles areproduced without the use of the solvents needed for an emulsionpolymerization process, and the process yields particles of narrowersize distribution.

The problem of water solubility during the collection in water ofspherical particles produced from agarose has been avoided in U.S. Pat.No. 5,053,332 by allowing the droplets—until they are gelled—to fallthrough and remain suspended in an upward flowing cooling gas which isinert to agarose. Such a procedure has a desiccating effect on theparticles.

The purpose of the invention is to avoid the above-mentioned drawbacksof the known technique by producing an improved porous sphericalmaterial which preferably is intended for chromatography.

For water soluble polymeric materials the high Is demands that organicsolvents must not be present in the material intended for chromatographyare eliminated by the invention. Furthermore, new processes are obtainedby the invention, in which organic solvents can be avoided, an aspectwhich is important from an environmental point of view.

More specifically, the invention refers to a method of producing porousspherical particles from a polysaccharide in solution, which can begelled and/or polymerized, the polysaccharide solution being finelydivided by mechanical means into spherical droplets. The droplets areallowed to pass through a controlled atmosphere, and the finely dividedmaterial formed is then allowed to pass (e.g. fall down) into acapturing medium. In this way, spherical particles having a narrowparticle size distribution can be continuously produced from apolysaccharide.

Such particles can for example be achieved with the device described inWO 88/07414. With this system, droplets having a diameter of 20-500 μmcan be produced.

The method according to the invention is based on the fact that when apolysaccharide, which can be gelled, is formed into a droplet, thedroplet under certain circumstances is gelled into a three dimensionallattice enclosing a solvent in such a way that a pseudosolid material isformed. Thus, the molecular structure in the droplet formed has—from thevery beginning—defined the maximal porosity of a future particle. Thestructure determining the maximal porosity is initially created in adroplet and the final pore formation is the result of a combination witha subsequent partly irreversible evaporation and desiccation process.

Accordingly, it is very important that the pore formation in thedroplets takes place under controlled conditions during the gelling ofthe polysaccharide. If the evaporation or the desiccation is tooextensive, the surface of the future particles will dry and an irregularsurface will be obtained the porosity at the same time beingconsiderably affected. The risk of an uncontrolled desiccation increaseswhen a smaller particle size is contemplated.

The controlled atmosphere is according to the invention a humidatmosphere, the temperature and humidity of which is controlled. Porousspherical particles of retained or increased porosity are obtained aftera conveyance through an atmosphere, which thus consists of a varyingmixture of air and water in gaseous phase or water vapor only. Theatmosphere used is also controlled by its temperature. A temperaturegradient will inevitably be obtained, but it can be controlled.Preferably, the temperature gradient is controlled to be as smooth aspossible from the site of droplet formation to the capturing medium.

After this controlled conveyance through a humid atmosphere, thepolysaccharide droplets are captured. In the capturing medium, thegelling of the droplets/particles is completed. Preferably, thecapturing medium comprises water, but it can also be an organic solvente.g. toluene, when a low surface tension is of importance for themanufacturing of completely spherical particles.

The porous spherical particles formed can then be separated by means offiltration or sedimentation.

The droplets from a particle generating apparatus contain apolysaccharide solution, which can be gelled, and the gelling may or maynot be continues during the conveyance through the controlledatmosphere. Thus, the temperature of the humid atmosphere is higher thanthe gelling temperature of the polysaccharide. The controlled atmospherecan be of the same temperature as the environment, for example roomtemperature, but higher as well as lower temperatures may be used independence of the application contemplated. Accordingly, thepolymerization of a material, which can be gelled, can also becontrolled to take place during the conveyance through the controlledatmosphere. The residence time in this atmosphere is usually less thanabout 30 seconds, but can of course be varied.

This procedure results in that the surface porosity of the newly formedparticles is maintained throughout the gelling stage. In thisconnection, a desiccation of the particle surface is avoided during thegelling, which would result in an extensive reduction of the porosity.

In principle, the polysaccharide used for exercising the methodaccording to the invention can be any naturally occurringpolysaccharide. Preferably, the polysaccharide is agarose, agar, starch,or alginate.

The polysaccharide is dissolved in a solvent comprising water. If thegelling polysaccharide is agarose, it is made as water solution of 2-14weight %. In this case, the gelling process takes place at a highrelative humidity and at a high temperature. Preferably, the cooling ofthe droplet takes place in a water vapor (100% relative humidity). Thedroplets obtained are conveyed to a capturing medium through anatmosphere controlled with respect to humidity and temperature, whichpreferably consists of a mixture of air and water vapor, alternativelywater vapor only. During the conveyance, the droplets are subjected to atemperature gradient, preferably between 100° C. and 20° C., thesteepness of which can be controlled. The final gelling can also beachieved in the capturing medium.

The polysaccharide gel solution as well as the aqueous capturing mediumcan advantageously also contain additives affecting the gelling, such assalts etc. Since the polysaccharide is substantially soluble in water, acomplete gelling at low temperature can be effected in the aqueouscapturing medium. The capturing medium can also contain a surfacetension reducing agent in order to facilitate the transport through thesurface of the medium.

EXAMPLES

The following non-limiting examples will now be given in order tofurther describe the invention.

Example 1

Agarose was dissolved to 4 weight % in boiling water, and the solutionwas then allowed to cool to 90° C. while stirring. At this temperaturethe agarose solution obtained was finely divided into spherical dropletsof 100 μm±25 μm by means of the device described in WO 88/07414. Then,for about 30 seconds the spherical droplets obtained were allowed tofall through a zone of water vapor/air of a high moisture content(50-100%) and temperature (20-100° C.). The particles were then allowedto fall down into a tank containing water of room temperature, theparticles being completely gelled therein. The preparation of oneseparate batch for chromatography results in a particle sizedistribution of more than 80% of the material within ±25% of the meanparticle size.

Example 2

Agarose particles were produced by disperging 6.6 g of agarose powder to100 ml of water having a temperature of about 30° C. The agarose powdersuspension was then completely dissolved with stirring in a microwaveoven at about 96° C.

The gel solution obtained was sprayed by means of a rotating disk intoindividual droplets, as shown in WO 88/07414. The droplets formed werecaught in water, which resulted particles having a diameter of 100μm±10% and a dry weight of about 6% (w/v).

Before being captured, the droplets formed by the rotating disk wereallowed to pass through a controlled atmosphere according to theinvention. This was accomplished by arranging a dome over the spinningdisk and controlling temperature and humidity underneath to be 50° C.and 100%, respectively.

As a comparison, the droplets formed were allowed to pass normal roomatmospheric conditions, i.e. a temperature of 20° C. and a humidity of55%.

The porosities of the particles formed were compared after passagethrough the different atmospheres and transfer to a capturing medium.This was performed in column (Ø=10×h=300 mm) which was equilibrated andeluted with 25 mM Tris-HCl, pH 7.0, and 100 mM KCl at a rate of 15 ml/h.The column was loaded with different substances of known molecularweights. Any appearance of a substance after the void volume was noted(Yes), which indicated diffusion of he substance into the porousspherical particles. The results are given in Table 1 below.

TABLE 1 Substance Mw (D) Comparison Invention Thyroglob. 669 000 0 YesFerritin 440 000 0 Yes Catalase 232 000 Yes Yes Aldolase 158 000 Yes Yes

The results show that particles produced according to the invention haveporosities that allow diffusion of substances of very large molecularweights, up to more than 650 000 Dalton. Particles under normalatmospheric conditions resulted in less porous particles. They onlyallow diffusion of considerably smaller substances, i.e. less than 250000 Dalton. Consequently, particles produced according to the inventiondemonstrate increased porosity.

1. In a method of controlling the porosity of porous spherical particlesproduced from a polysaccharide dissolved in a solvent including water,wherein the polysaccharide solution is finely divided by mechanicalmeans into spherical droplets and transferred to a capturing medium, theimprovement comprising conveying said droplets through a humidatmosphere, wherein the temperature and, humidity of said atmosphere arecontrolled.
 2. The method of claim 1, wherein said temperature and/orsaid humidity of said atmosphere is set above ambient temperature and/orhumidity, and said porosity is retained or increased.
 3. The method ofclaim 2, wherein the temperature of said capturing medium is broughtbelow that of the gelling temperature of the polysaccharide.
 4. Themethod of claim 1, wherein said capturing medium comprises water.
 5. Themethod of claim 1, wherein said polysaccharide is selected from thegroup comprising agarose, cellulose, starch, and alginate.
 6. The methodof claim 5, wherein said polysaccharide is agarose and is dissolved as a2-14 weight % water solution.